【作者】 敖宁；

【导师】 刘玉琴；

【作者基本信息】 北京协和医学院 ， 病理学与病理生理学， 2014， 博士

【摘要】 泛素特异肽酶22(ubiquitin-specific processing peptidase22, USP22)为hSAGA (human Spt-Ada-Gcn5-acetyltransferase)转录辅助因子复合物的亚基,通过对组蛋白尾部的特定氨基酸去泛素化修饰使转录复合物进入到目标基因启动子上,激活基因转录。USP22作用广泛,主要包括三大类功能：一,USP22去泛素化相关蛋白,包括SIRT1、 FUBP1、 IRF1、 TRF1等。USP22通过与这些蛋白作用进一步调控其下游靶基因/蛋白。二,USP22直接激活一些基因的转录。ChIP证明USP22与Myc靶基因CDCA7、 CyclinD2、 ODC、CAD、 MTA1的启动子区结合并激活这些基因转录。三,USP22直接或间接与一些基因的表达相关,包括BMI-1、 c-Myc、 pAkt、 p16INK4a、 p14ARF、 CyclinD2、 p53、 p21等。一组包含11个基因的癌死亡标签(death-from-cancer signature)的高度表达能够鉴别出有治疗耐药、复发和远处转移等特性的恶性肿瘤细胞,USP22为其中之一。USP22促进多种肿瘤细胞系增殖、抑制细胞凋亡,并且通过去泛素化SIRTl抑制结肠癌细胞凋亡。本研究探讨USP22对STAT3的影响及其对结肠癌细胞侵袭能力的作用。首先,本研究在模式工具细胞293T中转染四种表达质粒的不同组合,在强化表达体系(不同蛋白有足够多的量来研究蛋白间相互作用)中通过免疫共沉淀、Western Blotting验证了USP22去泛素化SIRT1、SIRT1去乙酰化STAT3; Western Blotting证实USP22可借助SIRT1降低STAT3乙酰化水平。接下来,本研究检测了6株结肠癌细胞系中USP22的表达,选用高表达USP22的结肠癌细胞HCT116、 HT29、SW480,其中结肠癌细胞系HT29、 SW480中通过免疫共沉淀、Western blotting等方法研究USP22和SIRT1、STAT3之间的关系,发现抗USP22抗体免疫沉淀的蛋白中可检测到STAT3,表明USP22与STAT3存在内源性相互作用。同时,HT29、 SW480细胞中用强化表达体系发现过表达SIRT1时,USP22与STAT3之间的作用增强,说明USP22、 SIRT1、 STAT3三种蛋白以复合体形式存在。HT29、 SW480细胞中USP22同样通过SIRT1降低STAT3乙酰化水平,这阐明了USP22/SIRT1/STAT3之间存在的表观遗传调控方式。其次,本研究用siRNA干扰技术在体外有效敲低USP22在结肠癌细胞HT29、SW480中的表达,发现SIRT1表达下调、STAT3的乙酰化水平升高、STAT3促进肿瘤侵袭方面的靶基因TWIST、MMP9表达升高,而STAT3mRNA和蛋白水平并没有改变,说明USP22通过去泛素化SIRT1这种表观遗传机制下调STAT3乙酰化水平、影响STAT3的转录活性。同时,Real Time PCR、Transwell细胞侵袭实验等证实HT29、 SW480细胞中联合敲低USP22/STAT3之后TWIST/MMP9mRNA水平、细胞体外侵袭能力较单独敲低STAT3明显升高。因此,本研究首次证明了USP22可通过SIRT1/STAT3/MMP9通路影响结肠癌细胞的侵袭能力。此外,39例结肠癌组织芯片中检测USP22的表达与STAT3表达(总蛋白)成中度正相关(r=0.457P<0.01),与MMP9表达成低度正相关(r=0.385P<0.05)而STAT3表达与MMP9表达高度相关(r=0.785,P<0.01)。同时我们在结肠癌侵袭前沿观察到USP22低表达、MMP9滴状高表达,说明USP22表达下降可负向调控MMP9表达以及结肠癌的侵袭,这与我们体外研究的结果一致。总之,我们的研究首次揭示了USP22与SIRT1、 STAT3形成蛋白复合体,并且USP22通过SIRT1/STAT3/MMP9途径抑制结肠癌细胞的侵袭,本研究加深了USP22生物学功能的理解以及USP22在结肠癌细胞侵袭中作用的认识。更多还原

【Abstract】 USP22(ubiquitin-specific processing peptidase22) is a subunit of hSAGA (human Spt-Ada-Gcn5-acetyltransferase) co-activator complex. Within hSAGA, USP22deubiquitylates histone H2A and H2B to regulate the transcription and expression of the related genes. USP22is involved in three categories of cellular functions. First, deubiquitination of related proteins, such as SIRT1, FUBP1, IRF1, TRF1. Second, transcriptional regulation of specific genes. USP22is recruited to Myc target genes CDCA7, CyclinD2, ODC, CAD, MTA1by Myc oncoprotein. Third, USP22is related with expression of some genes directly or indirectly, such as BMI-1, c-Myc, pAkt, p16INK4a, p14ARF, CyclinD2, p53, p21. Now that it is believed that USP22is one of the death-from-cancer signature and associated with proliferation, distant metastasis and poor prognosis. USP22could promote tumor proliferation and suppress tumor cell apoptosis. USP22de-ubiquitinates SIRT1to suppress colon cancer cell apoptosis. In this research, we report a new mechanism by which USP22affects SIRT1-STAT3pathway and colon cancer invasion.First, to explore protein-protein interaction,293T cells were employed as cellular model with force-rexpressed system (assuring there is enough amount of proteins) to observe protein-protein interaction. As was proved, USP22could deubiquitinate SIRT1and SIRT1down regulates STAT3acetylation, four plasmids in different combination were transfected in293T cells to certify the USP22-SIRT1-STAT3interaction. We found USP22could down regulate STAT3acetylation through SIRT1. Then we screened USP22expression in6colon cancer cells and HT29, SW480cells with high-expression of USP22were picked up for the following research. To demonstrate USP22-SIRT1-STAT3interaction, co-immuno-precipitation and western blotting were used again. STAT3interaction with USP22was shown by anti-USP22antibody pull down and the detection of STAT3using an anti-STAT3antibody in HT29, SW480cells, suggesting that endogenous USP22 interacts with endogenous STAT3. The interaction of USP22and STAT3was strengthened in SIRT1-overexpressed cells, suggesting USP22interacts with STAT3mediated by SIRT1and USP22, STAT3, SIRT1are also present in a single protein complex. Then we found USP22could down regulate STAT3acetylation through SIRT1in HT29, SW480cells. Therefore our research demostrated the presence of USP22/SIRT1/STAT3pahtway and clarified the epigenetic mechanism of their interactionSecond, depletion of endogenous USP22by RNA interference in HT29, SW480cells destabilized SIRT1and elevated STAT3acetylation. USP22knockdown also promoted expression of STAT3target genes MMP9and TWIST, which are involved in tumor invasion, but STAT3expression was not affected, confirming USP22antagonizes the STAT3transcription activation by deacetylation of STAT3at posttranscriptional level. USP22/STAT3co-depletion partly rescued reduced MMP9and TWIST mRNA levels as well as cell invasion, induced by STAT3depletion alone, which were tested by Real Time PCR and Transwell assay. Therefore, for the first time, we identified USP22affects colon cancer cell invasion by SIRT1/STAT3/MMP9pathway. USP22expression positively correlated with STAT3expression (r=0.457, P<0.01) and MMP9expression (r=0.385, P＜0.05); STAT3expression positively correlated with MMP9expression (r=0.785, P＜0.01) in colon cancer TMA including39patients. In the mean time, we observed that USP22expression was lower in the leading area of colon caner invasion compared to colon cancer, but MMP9expression was higher, suggesting USP22may downregulate MMP9expression and colon cancer invasion.In conclusion, our study reveals USP22, SIRT1, STAT3are present in a single protein complex,and elucidates that USP22could suppress colon cancer cell invasion by SIRT1/STAT3/MMP9pathway. The research broaden our knowledge about the biological function of USP22and its role in colon cancer invasion.更多还原